The Nkx3. 1 homeobox gene is a key regulator of prostate epithelial differentiation whose loss-of-function represents an enabling event in prostate cancer initiation. Our proposed studies are based on the hypothesis that the molecular processes by which NkxS. 1 regulates prostate epithelial differentiation are causally linked to its role in cancer initiation, while its consequences for malignancy are limited by cellular senescence. We will utilize state-of-the-art molecular approaches applied to in vivo mouse models, organ transplant models, and cell-based approaches, and combined with validation to human prostate cancer to Identify key molecular pathways involved in prostate cancer initiation. Specifically: In Aim 1, we will investigate the relationship between cellular senescence and cancer initiation. Based on our observation that Nkx3.1 mutant mice display senescence coincident with the occurrence of PIN (Preliminary Data), we will investigate the relationship of the senescence phenotype to cell type, and prostate stem cells. We will evaluate the functions of senescence modulators in renal graft assays and validate their relevance to human prostate cancer with assistance of Core A. In Aim 2, we will investigate the relationship between cellular specification and cancer initiation. Based on our finding that gain-of-function of Nkx3.1 in nonprostatic epithelium is sufficient to induce prostate growth in vivo (Preliminary Data), we will now investigate the mechanisms by which Nkx3.1 induces prostate specification and their relationship to cancer initiation. To do so, we will use gene expression profiling followed by functional analyses of selected genes, which will be prioritized in conjunction with the bioinformatic component of Core B and validated to human prostate cancer with Core A. In Aim 3, we will elucidate a transcriptional program of cancer initiation. Having identified putative target genes that are relevant for cancer initiation and are both regulated by (i.e., from gene expression profiling) and bound (i.e., from chromatin immunoprecipitation, ChIP) by Nkx3.1 (Preliminary data), we will now pursue a comprehensive analysis of Nkx3.1 target genes using gene expression profiling combined with high-throughput genome-wide sequencing of ChlP-enriched DNA fragments (ChlP-Seq). Candidate target genes will be prioritized with assistance from the bioinformatic component of Core B and based on their expression in human prostate cancer with Core A. In addition to essential interactions with Cores A and B, our studies will benefit from interactions with Michael Shen (Project 1) to evaluate the molecular properties of senescence in the context of prostate stem cells, and with Edward Gelmann (Project 3), who will provide an in-depth evaluation of the consequences of Nkx3.1 for DNA damage.